Glucose is one of the most important biochemical substrates for cellular catabolism. Glucose biosensors measure glucose based on enzymatic recognition of glucose by glucose oxidase (GOx) and have been widely used in diagnostics (e.g. diabetes monitoring) and research (e.g. β cell physiology study).
ATP is the fundamental unit of currency in cellular energetics, and also an important extracellular messenger in eukaryotic systems. Biosensors have been developed to measure ATP which show great advantages over conventional techniques. The main difference between ATP biosensors and glucose biosensors is that ATP biosensing is based on a multi-enzyme approach to convert ATP into an electro-oxidative species. ATP can be measured by combining hexokinase and GOx. One major drawback with this scheme is that the linear range is up to 200 nM, which is lower than human plasma ATP concentration (up to 11 μM) and limits its physiological applications. A second measurement scheme using a two enzyme system of glycerol kinase (GK) and glycerol-3-phosphate oxidase (G3POx) is adopted in this study, because it has been reported to have a wider linear range (up to 50 μM) compared with the first scheme, which is important for physiological applications. Additionally, it has been applied in vivo to study ATP signaling during spinal motor activity. Although ATP biosensors possess the potential to be an extremely useful tool for cell physiology, reports on electrochemical ATP biosensors are still quite limited.
Electrochemical biosensors are highly effective in monitoring biomolecule concentrations due to the high sensitivity, real-time monitoring capabilities and low cost. This contrasts with conventional measurement techniques including radioisotope tracing, NMR spectroscopy, and microfluorometry assays, which are complex and expensive but also are severely limited in terms of spatial and temporal resolution. Nanomaterials with good biocompatibility and electrocatalytic activities have received a lot of attention in terms of biosensing performance. However, biosensors based on conventional materials are constrained in terms of total sensitivity, which in turn limits the potential for miniaturization. This is due to restrictions in mass transport, enzyme loading, and electrochemical coupling. These sensitivity issues not impact the limit of detection, but also the signal-to-noise ratio in measuring very small changes in concentration over time. These parameters are key to exploring important physiological phenomena including β cell glucose consumption during insulin secretion.
Carbon nanotubes (CNT) and metal nanomaterials are the most commonly used nanomaterials in biosensor construction. CNTs are an allotrope of carbon. The carbon atoms at tube ends or at tube defect sites possess the catalytic capability for electrochemical reactions. The major obstacle for CNT immobilization is the fact that CNTs are minimally soluble in aqueous media due to van der Waals aggregation. Abrasive immobilization, CNT suspending polymers, linking agents and chemical vapor deposition (CVD) are the most successful current approaches for CNT preparation. Apart from these approaches, biochemical modification of CNTs (e.g. glucosamine and single-stranded DNA (ssDNA)) significantly increases the solubility in water, thus opening up technical approaches for CNTs that can be mediated in aqueous media. This greatly enhances the application of CNTs for microbiosensor applications. There are problems associated with current approaches. Abrasive immobilization is not possible with microscale devices. The main problem with polymers and linking agent immobilization is that residual materials remain on the biosensor after CNT immobilization, which limits mass analyte transport and sensor sensitivity. By adopting aqueous media based modifier approaches there is potential to apply CNT nanomaterials to microbiosensors using technical approaches that will preserve the effective surface area. Compared with other approaches, CVD is relatively complicated and expensive, ssDNA modification is much easier and cheaper. ssDNA modified single-walled CNTs have been demonstrated to dramatically increase the electroanalytical current output, while decreasing the redox overpotential for electrochemistry, and has been used as a catalyst for redox reactions. This is explained by virtue of a systematic change in SWCNT valence energy levels due to DNA wrapping. Sensors incorporating ssDNA-SWCNT without enzymes exhibited increased sensitivity towards the electrochemical measurement of dopamine via direct oxidation.
Platinum black (Pt black) is a metal formed by electrodepositing amorphous clusters of Pt nano-particles. Pt black has been used to enhance biosensor sensitivity due to the catalytic activity of Pt nano-particles, and the ease of attaching enzymes to Pt via cross-linking agents (e.g. glutaraldehyde). Previous studies that combine both CNTs and Pt nanomaterials have exhibited improved performance than using either material alone.